Haemocoagulase

ABSTRACT

The present invention provides a venene haemocoagulase gene and its expression for the functional protein. The haemocoagulase gene of the present invention has a nucleotide sequence shown by the sequence list SEQ ID NO: 1 or the mutated nucleotide sequence formed by replacement, depletion, or addition of one or more nucleotide based on the said nucleotide sequence with an equivalent function. The said haemocoagulase has amino acid sequence shown by SEQ ID NO: 2. Haemocoagulase of the present invention has an obvious hemostatic effect, a broad effective dose range, safe and reliable application, which creates good conditions for the development of genetic engineering products of recombinant haemocoagulase in the future.

TECHNICAL FIELD

The invention belongs to the field of biotechnology, specifically relates to a venene protein, haemocoagulase and its encoding gene.

BACKGROUND ART

Reptilase, also called haemocoagulase, is a novel haemostat drug with clinical application in recent years. It is an enzymatic haemostat isolated from the venom of fer-de-lance (Bothrops Atrox) and contains two active ingredients, batroxobin and clauden (a phospholipid-depending Factor X Acitivator). Batroxobin in reptilase stimulates the degradation of fibrinogen into fibinopeptide A and increased number of defibrination monomers which could linked to form fibrin I multimers. Haemocoagulase has an effect on the process of the cross-linked fibrin formation by fibrin I multimers, which results in the function of hemostasis. The fibrin I multimer can also stimulate platelet aggregation around vascular lesions and accelerate the formation of platelet tampon, thereby accelerating the effect of hemostasis at the site of vascular lesions. Clauden in reptilase can activate the Blood coagulation factor X which were concentrating on the surface of phospholipid reaction and then convert it into blood coagulation factor Xa. Factor Xa could then form a complex prothrombin activator together with calcium ion, blood coagulation factor Va and platelet phospholipid (PF3). This complex prothrombin activator catalyzes conversion of prothrombin to thrombin at the vascular lesions, thereby promoting the formation of blood clotting and thrombus. Reptilase does not induce blood clotting of the blood vessels with a usual dose. It has the effect of hemostasis only under the condition of bleeding, and it shows good therapeutic effect during the clinical application.

There are abundant research resources on snake venom in China. It has been reported that a special haemocoagulase with hemostasis effect can be isolated and purified from the snake venom of Agkistrodon acutus in China. However, due to the complicated components in the snake venom, there are two disadvantages of the biochemical venom products obtained by biochemical process: a) the whole operation is costly and difficult, b) other residue components after the process of purification may lead to different therapeutic effects and toxic side effects.

Therefore, genetic engineering was introduced into the study of batroxobin. The first application was in 1991. cDNA of batroxobin was isolated from the cDNA library of B. atroxmoojeni by Maeda etc., and was cloned into the expression vector of E. coli by recombination. Fusion proteins were expressed in the form of inclusion, and part of natural activities was obtained by refolding. However, the expression of mature and functional protein of batroxobin was not detected. In terms of structure, most batroxobins are glycoproteins. The expressed product can not be glycosylated due to the limit of the E. coli expression system itself, that is, lacking the function of posttranslational processing of proteins which is very important to protein intrinsic property and its biological functions. In 1996, Ancrod, a batroxobin originating from agkistroclon rlzodoston, was successfully expressed in epidermal cells of mice by Geyei etc. It was the first time that batroxobin was expressed in mammalian cells. It also showed that the structure of glycosylation is closer to that of the natural enzyme. Subsequently, batroxobin genes were cloned into pichia yeast to obtain a secreted product, Gussurobin, which also showed a higher biological activity. Therefore, it is generally believed that the expression of batroxobin genes should be performed inside the eucaryotic expression system.

CONTENT OF THE INVENTION

The object of the present invention is to obtain and provide a valid haemocoagulase with good haemostatic effect from Agkistrodon acutus, as well as its encoding genes.

The nucleotide sequence of haemocoagulase gene of Agkistrodon acutus according to the present invention (which will now be referred to as SL32 gene) is shown by sequence list SEQ ID NO: 1. The amino acid sequence of protein encoded by above-mentioned gene is shown by sequence list SEQ ID NO: 2. The present invention further includes the amino acid sequences that are formed by replacing, depleting, or adding one or more amino acids according to the amino acid sequence shown by SEQ ID NO: 2 and possess an equivalent function and their encoding nucleotide sequences.

There is a high homology between the SL32 gene and mRNA of batroxobin (gi:211023. SL32 gene has 1326 base pairs, and the mRNA of batroxobin has 1504 base pairs. SL32 encodes the protein with 255 amino acids, batroxobin also has 255 amino acids. The difference between them is that the eleventh N-terminal amino acid is glutamine (Q) in SL32, while it is leucine (L) in the primary structure of batroxobin.

Although proteins encoded by the SL32 gene seems to be very similar to batroxobin, they have significantly different activity due to their different origins and definitely different nucleic acid sequence and primary structure of amino acids. The 30 amino acids at N-terminus of SL32 encoding batroxobin-like amino acid sequence is as follows:

(SEQ ID NO: 3) Met-Val-Leu-Ile-Arg-Val-Ile-Ala-Asn-Leu-Gln-Ile- Leu-Gln-Val-Ser-Thr-Tyr-Ala-Gln-Lys-Ser-Ser-Glu- Leu-Val-Ile-Gly-Gly-Asp.

The assays showed that SL32 had a significantly better ability to hydrolyze the metaglobulin Aαstrand than batroxobin.

The present invention obtained the haemocoagulase by the process of isolating and purifying the venom of Agkistrodon acutus and further obtained its encoding genes. More particularly, the technical solution of the present invention is as follows: isolating and purifying haemocoagulase from snake venom of Agkistrodon acutus, sequencing its N-terminal amino acid sequence, designing and synthesizing the oligonucleotide probes according to the sequence, extracting mRNA from toxic glandular tissues of the Agkistrodon acutus, reversely transcripting mRNA and constructing the cDNA library, screening the cDNA library with the probes above and performing sequencing analysis of the positive clones respectively, and then cloning the target genes which were screened out into the expression vector. (see FIG. 1)

1. Isolation and Purification of Haemocoagulase

Active ingredients with fibrinogen aggregation activity were isolated and concentrated directly from snake venom by using the DEAE Sepharose Fast Flow column, manufactured by GE Healthcare, and isolated by gel filtration chromatography using the Sephacryl S-200HR column, then further collected and purified by using the DEAE 8HR HPLC column manufactured by Waters Corp.

2. Identification of the N-terminal Amino Acid Sequence and Synthesis of the Oligonucleotide Probe

The isolated and purified protein mentioned above was performed SDS-PAGE electrophoresis, then transferred to the PVDF memberane. The strip corresponding to the protein was cut and the N-terminal of the peptide chain was determined by Edman degradation using the 120-A gaseous phase sequenator of the American ABI company. According to the sequencing result, the possible nucleotide sequence was deducted and the sequence of the probe is tggtcattggaggtgatgaa (SEQ ID NO: 4). The corresponding oligonucleotide probe was synthesized by using the ABI 3900 gene synthesis machine.

3. Construction of cDNA Library

1 g fresh Agkistrodon acutus glandular tissue was taken and the total RNAs of sample cells were extracted by using the Qiagen RNeasy mini kit, and mRNA was then purified and obtained from the aforementioned total RNAs by using the mRNA Purification Kit. The cDNA was further synthesized by the TimeSaver™ cDNA Synthesis Kit of GE Healthcare. Then both ends of the cDNA were ligated with EcoRI/NotI adapters, and the cDNA with adapters was purified through SizeSep™ 400 Spun Columns of the company. Then the purified cDNA was cloned to a λgt11 vector manufactured by Promega Company by the double enzyme digestion method. After being transformed to E. coli, the experiment was followed by amplification culture, clonal and titre identification.

4. Selection of Positive Clone

The total λDNAs of the cDNA library mentioned above were extracted as a template, the synthesized N-terminal primers (tggtcattggaggtgatgaa (SEQ ID NO: 4)) were labeled with P³² by using the HexaLabel™ DNA Labeling Kit made by GE Healthcare. The probe was used for in situ hybridization screen of the cDNA library and the positive bacteria colonies were then selected out.

5. Acquisition of Complete Sequence of Gene Encoding Haemocoagulase Protein

λDNA was extracted from these positive colonies mentioned above after amplification culture, then sequenced with the primers (forward: GGTGGCGACGACTCCTGGAGCCCG (SEQ ID NO: 5), reverse: TTGACACCAGACC AACTGGTAATG (SEQ ID NO: 6)) on plasmid using 3730 Gene sequence analysis of ABI.

6. Construction of Expression Vector, Transformation of the E. coli, Identification of the Positive Clones

A primer pair (Forward atggtgctgatcagagtgatagca (SEQ ID NO: 7); Reverse cgggcaagtcgcagttttatt (SEQ ID NO: 8)) was designed to ampilfy ORF of the above-mentioned haemocoagulase.

Amplified product was cloned into the E. coli expression vector pET31b(+) made by Novagen company . Then the expression vecctor was transformed into E. coli host Rosetta (DE3) pLysS, followed with identifying and selecting transformed positive clones by PCR prior to further sequencing identification.

7. Expression of Target Genes and Acquisition of Proteins

The proteins expressed in bacteria were extracted with the BugBuster kit made by Novagen. Studies shows that the mixed solution of 250 mM of NaCl, cyclode-xtrin, GSH/GSSG, EDTA can most effectively promote and produce the SL32 proteins with biological activity among total 96 condition combinations.

Of course, there are some other methods for obtaining the complete sequence of the gene encoding the haemocoagulase, for example, designing and synthesizing upstream and downstream oligonucleotide primers according to the indentified N-terminal amino acid sequence as well as the homology sequences of other snakes. The total mRNA obtained from crotalin glands of Agkistrodon acutus could be used as a template and cDNA could be obtained by reverse transcription, followed by sequencing and designing to synthesize related primers, thereby further cloning the complete gene sequence.

The invention makes use of the unique resources of Agkistrodon acutus in china, to isolate haemocoagulase from the snake venom, and further to obtain the genes of haemocoagulase by cloning, which creates good conditions for the development of genetic engineering products of recombinant haemocoagulase in the future.

DRAWINGS OF INVENTION

FIG. 1 is a flow diagram of SL32 gene cloning

FIG. 2 is a SDS-PAGE analysis electrophoresis picture of hydrolysis of fibrinogen by SL32 and Reptilase, wherein T represents human thrombin, H represents SL32, F represents fibrinogen control, R represents Reptilase, the numbers after these letters represent incubation time (hour), and M represents standard molecular weight.

MODE OF CARRYING OUT THE INVENTION

The present invention will be further illustrated with reference to the examples as follows, but the scope of the present invention is not limited thereto.

EXAMPLE 1 Isolation and Purification of Haemocoagulase

The collected snake venom was dissolved in 0.02 M of Tris-HCl buffer solution with a pH of 8.0 and centrifuged for thirty minutes. The DEAE Sepharose Fast Flow column manufactured by GE Healthcare was balanced with the buffer solution above, to which the supernatent obtained by centrifugation was transferred. The elution was conducted with a linear concentration gradient of NaCl with an eluting temperature of 4° C. and a flow rate of 80 mL/h (starting from a balanced concentration until a concentration of 0.02 M of Tris-HCl, 0.2 M of NaCl and a pH of 8.0). The eluted proteins with respective peak value were collected and the fibrinogen aggregation activity was tested. It has been found that the resulting protein peak eluted at 0.02˜0.08 M of NaCl concentration has fibrinogen aggregation activity. The active ingredients were applied to the Sephacryl S-200HR column and isolated by gel filtration chromatography. The elution solution was 0.02 M of the Tris-HCl buffer solution with a pH value of 8.0. The ingredients with fibrinogen aggregation activity (primary eluting peak) were collected and further isolated by the DEAE 8HR HPLC column (The column had been balanced with 0.02 M of Tris-HCl buffer solution with a pH of 8.0 beforehand) manufactured by Waters Corp. The elution solution was the same as the elution solution for DEAE Sepharose Fast Flow column; the protein peak eluted with 0.03-0.06M NaCl solution has fibrinogen aggregation activity; and the purified fractions with fibrinogen aggregation activity (primary eluting peak) were collected.

EXAMPLE 2 Identification of the N-terminal Amino Acid Sequence and Synthesis of the Oligonucleotide Probe

The isolated and purified protein above was taken and transferred to the PVDF membrane after SDS-PAGE electrophoresis. The strip corresponding to the protein was cut and the N-terminal of the peptide chain was determined by Edman degradation using 120-A gaseous phase sequenator made by ABI. The N-terminal batroxobin-like 30 amino acid sequence of SL32 was as follows: Met-Val-Leu-Ile-Arg-Val-Ile-Ala-Asn-Leu-Gln-Ile-Leu-Gln-Val-Ser-Thr-Tyr-Ala-Gln-Lys-Ser-Ser-Glu-Leu-Val-Ile-Gly-Gly-Asp (SEQ ID NO: 3). According to the sequencing result , the potential nucleotide sequences were deducted, the hybridization probe was designed on-line by using Primer 3 software, the sequence of the probe was tggtcattggaggtgatgaa (SEQ ID NO: 4), and the corresponding oligonucleotide probe was synthesized by using the ABI 3900 gene synthesis machine.

EXAMPLE 3 Construction of cDNA Library

1 g fresh fresh Agkistrodon acutus tissue was taken; the total RNAs of sample cells were extracted using the Qiagen RNeasy mini kit. The mRNA was purified and obtained from the aforementioned total RNAs by using the mRNA Purification Kit. The cDNA was further synthesized by using of the TimeSaver™ cDNA Synthesis Kit made by GE Healthcare, ligated by EcoRI/NotI adapters at their both ends. The cDNA with adapters were purified by the SizeSep™ 400 Spun Columns of GE Healthcare Company and cloned into the λgt11 vectors manufactured by Promega Company using double enzyme digestion method. The recombinant plasmid was transformed into E coli then followed by amplification culture, clone identification and titer assay.

EXAMPLE 4 Selection of Positive Clone

The total DNA of the cDNA library mentioned above was extracted as a template, the synthesized N-terminal primers mentioned above (tggtcattggaggtgatgaa (SEQ ID NO: 4)) were labeled with P³² by using the HexaLabel™ DNA Labeling Kit of GE Healthcare. The probe was used for in situ hybridization screen of the cDNA library and then the positive colonies were selected.

EXAMPLE 5 Acquisition of Complete Sequence of Gene Encoding Haemocoagulase

λDNA was extracted from aforesaid positive colonies after amplification culture, then sequenced with the primer (forward:GGTGGCGACGACTCCTGGAGCCCG (SEQ ID NO: 5), reverse: TTGACA CCAGACCAACTGGTAATG (SEQ ID NO: 6)) on plasmid using the 3730 Gene sequence analysis made by ABI.

EXAMPLE 6 Construction of Expression Vector, Transformation of E. coli, Identification of the Positive Clones

A primer Pair (Forward:TCCCCTCTAGAATGGTGCTGATCAGAGTGATAGCAA (SEQ ID NO: 9); Reverse: GGTGCTCGAGTCACGGGCAAGTCGCAGT (SEQ ID NO: 10)) were designed to amplify ORF of the above-metioned haemocoagulase using high fidelity Taq enzyme, the condition of amplification reaction was at 96° C. 2 min, (at 96° C. for 30 s, at 59° C. for 30 s, at 72° C. for 1 min) total 25 cycles, at 72° C. for 7 min, store at 4° C. Xba I and XhoI were used to digest amplified products. Then the digested product was cloned to E. coli expression vector pET31b(+) (Novagen company). The recombinant plasmids were transformed into Ecoli host Rosetta (DE3) pLysS , followed by indentifying and selecting transformed positive clones by PCR prior to further sequencing identification, the sequencing primers are vector self-primers.

(SEQ ID NO: 11) T7 promoter: TAATACGACTCACTATAG (SEQ ID NO: 12) T7 terminator: GCTAGTTATTGCTCAGCGGT

EXAMPLE 7 Expression of Target Genes and Acquisition of Proteins

The screened positive clones with encoding mentioned above protein sequences were amplified and cultured respectively. The E coli proteins (inclusion body) were extracted using the BugBuster kit made by Novagen. The iFOLD™ Protein Refolding System 1 for the study of Protein Refolding from Novagen was further used to test the optimum condition for forming the spatial structure of the protein. Studies have found that the mixture solution of 250 mM NaCl, cyclode-xtrin, GSH/GSSG, EDTA can most effectively stimulate to form SL32 proteins with the biological activity among all 96 condition combinations.

EXAMPLE 8 Analysis and Identification of the SL32 Gene and Its Protein

BLAST result showed that SL32 gene has the highest homology with mRNA of batroxobin (gi:211023). The mRNA sequence of batroxobin has 1504 base pairs, while the SL32 gene whose CDS is (60) . . . (827), has 1326 base pairs altogether. Both SL32 and batroxobin have 255 amino acids. The difference between them lies in that the eleventh N-terminal amino acid is glutamine (Q) in SL32, while it is leucine (L) in the primary structure of batroxobin. Accordingly, the cloned SL32 gene is a wholly novel Haemocoagulase gene.

EXAMPLE 9 Functional Analysis of SL32 Expression Protein

Studies on the pharmacodynamic index, such as the blood coagulation system and the fibrinolytic system have been performed with Japanese albino rabbits and mice. The results showed that:

-   (1) SL32 has an effect on significant reduction of bleeding time. An     intravenous injection of 0.50 , 0.25 and 0.125 U/kg of SL32 protein     significantly shorten the bleeding time of the mice's cutted tails. -   (2) SL32 has an effect on stimulating blood coagulation. An     intravenous injection of 0.12, 0.06 and 0.03 U/kg of SL32 protein     significantly shorten the whole blood clotting time in the rabbits.     The effect is shown 10 minutes after administration; the peak level     is reached after half an hour; and the effect lasts for more than 6     hours. The difference between SL32 and positive control drug     Reptilase is not significant for the time and the strength of     stimulating blood coagulation. -   (3) For the allergic test of systemic administration of SL32, SL32     sensitized contact test and provocative contact test on guinea pigs     have been observed. No allergy has been found during 28 days     observation and the result of allergic test is negative. -   (4) For SL32 hemolytic test, no hemolysis and erythrocyte     aggregation have been observed from the hemolytic test on rabbit     blood in vitro. The result of hemolysis test is negative. -   (5) For SL32 vascular stimulation test, administration of the drug     by injecting rabbit at ear conch, ear edge vein and quadriceps     femoris in order to observe whether SL32 stimulates the     above-mentioned tissues or not. The result showed: injection of the     product does not cause red swelling, bleeding, denaturation or     necrosis of tissues; administration by the routes above is not     irritative; and the drug can be administered intravenously or     intramuscularly. -   (6) For SL32 nervous toxicity, bleeding toxicity and abnormal     toxicity tests, the mice, which were intravenously injected SL32,     were observed for the nevous toxicity and abnormal toxicity. The     mice, which were subcutaneously injected, were observed for bleeding     toxicity. The results of the test are negative in the range of     observed dosage. -   (7) For SL32 acute toxicity test, no adverse reaction has been     observed on either rats or mice injected via vena caudalis and     muscle once at 50 U/kg (equivalent to 3000 times of the clinical     human conventional dose). -   (8) For SL32 long term toxicity test, intravenously injected rats     with the drug at a dose of 1.36 u, 0.68 u or 0.34 u/kg, no toxic     effect has been found in total 22 tissue organs, including the main     metabolism and detoxification organs such as kidney, etc., and     central nervous system and heart, etc. -   (9) Under a condition with an equivalent activity, the SDS-PAGE     analysis of hydrolyzed fibrinogens with SL32 and Reptilase (see FIG.     2).

The electrophoretic result indicates that SL32 significantly hydrolyze fibrinogen Aα-strand and has a similar strength to human thrombin, while the hydrolysis of fibrinogen Aα-strand with Reptilase is still not evident. That is, SL32 is better than Reptilase in hydrolyzing fibrinogen Aα-strand.

The above results demonstrate that the expression product of SL32 has a significant hemostasis effect. Comparing with Reptilase, it has such features as a broader effective dose range, safer and more reliable application.

INDUSTRIAL APPLICABILITY

The present invention isolates and purifies haemocoagulase from Agkistrodon acutus venene and also finds out and clones its encoding genes. Haemocoagulase of the present invention has an obvious hemostatic effect, a broad effective dose, and safe and reliable application, which creates good conditions for the development of genetic engineering products of recombinant haemocoagulase in the future. 

1. An isolated venene heamocoagulase comprising SEQ ID NO: 2 or a sequence variant thereof, wherein said variant has heamocoagulase activity and is formed by replacement, deletion or addition of one or more amino acids of said SEQ ID NO: 2, with the exception of the amino acid at position 11 which is Gln.
 2. A hemostatic pharmaceutical composition comprising the venene haemocoagulase of claim
 1. 3. The venene hamocoagulase of claim 1, wherein the sequence variant includes insertion of a Thr between positions 16 and 17 of SEQ ID NO:
 2. 4. A venene haemocoagulase isolated from Agkistrodon acutus comprising amino acid sequence SEQ ID NO:
 2. 5. The venene haemocoagulase of claim 4, further comprising a sequence variant of SEQ ID NO: 2 formed by replacement, deletion, or addition of one or more amino acids of SEQ ID NO: 2, wherein the amino acid at position 11 of the variant of SEQ ID NO: 2 is Gln and wherein said variant possesses haemocoagulase activity.
 6. The venene haemocoagulase variant of claim 5, wherein the sequence variant includes insertion of a Thr between positions 16 and 17 of SEQ ID NO:
 2. 